This invention relates to polynucleotides and protein products encoded thereby. More specifically, the invention relates to polynucleotides of C. albicans and polypeptides encoded by the polynucleotides, which are associated with TUP1 gene fimction, particularly, gene repression and morphological transition, particularly to a filamentous form.
The yeast Candida is a ubiquitous human commensal, known as the causative agent of candidiasis. The majority of the diseases are caused by the species Candida albicans. It is the most prevalent commensal and opportunistic fungal pathogen of humans, causing common superficial infections as well as more serious systemic and organ infections. Cannon et al. (1995) J. Dental Research 74:1152-1161. Exposure to C. albicans at or shortly after birth results in lifelong colonization in the host tissues, such as the gastrointestinal tract, oral cavity and genital area. It has been noted that approximately 75% of women would suffer from vaginal candidiasis at some stage in their lifetime. Bossche et al. (1993) Fungal Dimorphism 3-10; Fidel et al. (1996) Clin. Micro. Rev. 9(3):335-348. Whereas C. albicans infection often remains localized to the initial sites of contact in healthy individuals, C. albicans cells can invade submucosal vessels, disseminate hematogenously and become life-threatening, especially to immunocompromised patients. The invasive forms of C. albicans infection are not only dangerous in their own right, but they are believed to facilitate infections by other opportunistic pathogens.
In the last decades, the incidence of severe and systemic candidiasis has increased dramatically because of the growing number of immunocompromised patients suffering from AIDS, diabetes, cancer and other conditions. In addition, the widespread use of immunosuppressants for organ transplant patients, the common practice of radiation and chemotherapy for treating malignancies, as well as the growing size of the aging population have increased the morbidity of this opportunistic pathogen. For reviews, see Rubin et al. (1993) Eur. J. Clin. Microbiol. Infect. Dis. 12 Suppl. 1, 542; Dudley et al. (1990) Pharmacotherapy 10:133; Paya (1993) Clin. Infect. Dis. 16:677-688; Rubin (1993) Eur. J. Clin. Micro. Infect. Dis. 12 Suppl. 1:S42-S48.
Despite decades of intensive study, the properties of C. albicans that contribute to its virulence are only beginning to be understood. Among the most investigated virulence factors are adherence, production of hydrolytic enzymes and adoption of various cell morphologies. Odds et al. (1994) Am. Soc. Microbiol. News 60:313-318. The ability of C. albicans to adhere to the host surfaces probably allows initial colonization and infection of the host tissues. Secretion of a variety of hydrolytic enzymes which are capable of degrading proteins and lipids is thought to generate tissue cavitation and thereby facilitate deeper penetration. The morphological transition between various forms of C. albicans is also considered a key determinant of virulence.
C. albicans cells can exist in a variety of shapes, ranging from ellipsoidal budding yeast cells (also known as blastospores) to filamentous forms in which cells remain attached to each other after dividing and thereby form long branched strings of connected cells. These filamentous forms include both pseudohyphae (where cells that form filaments are elongated, but still ellipsoidal) and true hyphae (where highly elongated cells that form the filaments are cylindrical and are separated by perpendicular septal walls). Transitions between the ellipsoidal and filamentous forms take place by outgrowth of new cells with the altered morphology, rather than remodeling of pre-existing cells. The ability of C. albicans to adopt these different morphologies is thought to allow the fungus to adapt to, and possibly travel to, different host micro-environments. Odds et al. (1988) Candida and Candidosis (Bailliere Tindall, London, 2nd ed.); Odds et al. (1994); Odds et al. (1994) J. Am. Acad. Dermatol. 31:52. The regulation of cellular morphology is in response to environmental conditions. In vitro studies have shown that most C. albicans strains assume filamentous forms when they are subjected to either unfavorable growth conditions, such as nutrient-poor media and high CO2:O2 ratio, or host-mimicking conditions, such as high temperature (37xc2x0 C.) and mammalian serum (10%). Conversely, rich media, low temperatures and aerated conditions promote blastospore growth. Intermediate conditions can induce various pseudohyphal forms as well as true hyphae. For reviews, see Odds et al. (1988) Candida and Candidosis, Bailliere Tindall, London, ed. 2nd; Odds et al Crit. Rev Microbiol. (1985) 12:45; Gow et al. (1984) Sabouraudia 22:137. Very little is known about the genetic identity of regulators controlling the morphological transition of C. albicans. 
The ability of C. albicans to adopt these different morphologies is thought to contribute to colonization and dissemination within host tissues and thereby to promote infection. Odds (1988); Odds (1994) J. Am. Acad. Dermatol. 31:S2. It has been commonly suggested that the hyphal form is invasive and pathogenic, while the blastospore is the commensal, non-pathogenic form. However, all morphological forms have been found within infected tissues. Histopathological examination of candidiasis lesions indicates that hyphae are not always present. More recent studies have shown that commensal C. albicans does not exist uniquely in the blastospore form. In fact, sometimes invading C. albicans cells are seen exclusively as the budding yeast form. Odds et al. (1994) Am. Soc. Microbiol. News 60:313-318. Despite the uncertainty with regard to the relative roles these two distinct forms of C. albicans have in fungal virulence, phenotypic switching represents a remarkable adaptation that C. albicans has acquired to cope with different host microenvironments.
The TUP1 gene of C. albicans appears to be a key regulator of filamentous growth. Braun et al. (1997) Science 277:105-109. Cells in which TUP1 function is disrupted grow exclusively as filaments in all conditions tested (e.g., nutrient-rich and nutrient-poor media, in the presence and absence of mammalian serum, aerobic and micro-aerobic conditions, throughout the range of temperature and pH values). These results suggest that the gene product Tup1 is a repressor of filamentous development. This function for the Tup1 protein may occur through transcriptional repression of genes whose expression is required to initiate and/or maintain filamentous growth. This conclusion is supported by: (1) the amino acid sequence of C. albicans Tup1 protein is very similar (67% identity) to that of the S. cerevisiae Tup1 protein, a known transcriptional repressor, and (2) expression of the C. albicans TUP1 gene is S. cerevisiae lacking TUP1 function restored wild type cell shape and growth behavior to the S. cerevisiae cells indicating that C. albicans TUP1 gene product functionally complements (i.e., substitutes for) S. cerevisiae tupl (i.e., lacking Tup1 fimction). For papers describing S. cerevisiae TUP1, see Tzamarias et al. (1994) Nature 369: 758; Komachi et al. Genes Dev. 8: 2857; Wahi et al. (1995) Genetics 140: 79-90; Edmondson et al. (1996) Genes Dev. 10: 1247.
Current therapy available for systemic candidiasis is limited to the use of anti-fungal agents. In practice, the arsenal of anti-fingal drugs is based on a few antimycotics, such as flucytosine, amphotericin B and azole derivatives. Many of these antimycotics are somewhat water insoluble which restrict their bioavailability and present problems in intravenous formulation. In addition, they cause serious and often difficult side effects, such as renal toxicity, bone marrow destruction, as well as unpleasant symptoms such as fever and shivering. Furthermore, the chronic use of these anti-fungal agents has led to the emergence of drug-resistant strains of Candida, which can cause fatal relapse of the disease. Dupont et al. (1995) J. Am. Podiatric Med. Assn. 85:104-115; Fox et al. (1991) J. Infect. Dis. 22:201-204; Scheife (1990) Pharmacotherapy 10:S133-S183. Taken together, anti-fungal therapy alone is inadequate for treating chronic candidiasis. The availability of recombinant cytokines, such as interleukin-2, provides an alternative way to stimulate the cell-mediated immunity of infected individuals. However, this type of cytokine replacement therapy for fungal infections remains highly experimental. Weinberg et al. (1990) N. Eng. J. Med. 332: 1718.
The transition between blastospore and filamentous forms may play a significant role in the pathogenicity of this organism. Mutant strains of C. albicans which failed to form filaments were found to be avirulent in a mouse infection model. Lo et al. (1997) Cell 90: 939-949. However, the C. albicans tup1 knockout mutant, which grows exclusively in filaments, is also poorly infective in mice. Braun et al. (1997); U.S. Ser. No. 60/051552. Identifying the genes regulated by TUP1, and thus that contribute to this morphological transition to growth in filaments and decreased infectivity, are therefore of great significance for identifying the role of this transition in pathogenesis and for developing potential therapeutic agents of candidiasis.
In view of the alarming prevalence of life-threatening candidiasis among immunocompromised patients and the lack of satisfactory agents to treat this condition, there is a pressing need for developing better therapeutic agents to combat C. albicans infections.
All publications cited herein are hereby incorporated by reference in their entirety.
This invention provides C. albicans RBT1 gene polynucleotide sequences, Rbt1 polypeptides encoded by these sequences, antibodies that bind to these polypeptides, compositions comprising any of the above, as well as methods using the polynucleotides, polypeptides, and/or antibodies.
Accordingly, in one aspect, the invention includes an isolated polynucleotide comprising a sequence encoding an Rbt1 polypeptide from C. albicans. The Rbt1 polypeptide encoded is found within the sequence depicted in SEQ ID NO:4 and embodiments included, including from about amino acid 1 to amino acid about 23, about amino acid 480 to about amino acid 496 of SEQ ID NO:4, as well as the sequence of SEQ ID NO:2, as well as the sequence of SEQ ID NO:4.
In another aspect, the invention provides isolated polynucleotides based on the sequence depicted in SEQ ID NO:3, and as such may comprise nucleotides from about 617 to about 685, from about 2054 to about 2104, from about 617 to about 2866 of SEQ ID NO:3, as well as the sequence of SEQ ID NO:1, as well as the sequence of SEQ ID NO:3.
In another aspect, the invention provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO:3. In another aspect, the isolated polynucleotide comprises a region of at least 20 contiguous nucleotides, with the region having at least 75%, preferably 85%, sequence identity with a sequence depicted in SEQ ID NO:3.
In another aspect, the invention provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1. In another aspect, the isolated polynucleotide comprises a region of at least 20 contiguous nucleotides, with the region having at least 75%, preferably 85%, sequence identity with a sequence depicted in SEQ ID NO:1.
In another aspect, the invention includes cloning vectors, expression vectors, host cells, and compositions comprising any of the above polynucleotides.
In another aspect, the invention provides an isolated polypeptide comprising an Rbt1 polypeptide sequence from C. albicans. The Rbt1 polypeptide(s) is found within the sequences depicted in SEQ ID NO:2 and SEQ ID NO:4, including from about amino acid 1 to about amino acid 23, about amino acid 480 to about amino acid 496 of SEQ ID NO:4, as well as the sequence of SEQ ID NO:2, as well as the sequence of SEQ ID NO:4.
In another aspect, the isolated polypeptide comprises a region of at least 10 contiguous amino acids which have at least 70% sequence identity to a sequence depicted in SEQ ID NO:4 wherein expression of said at least 10 amino acids is increased during conversion of C. albicans to filamentous form.
In another aspect, the isolated polypeptide comprises a region of at least 10 contiguous amino acids which have at least 70% sequence identity to a sequence depicted in SEQ ID NO:2 wherein expression of said at least 10 amino acids is increased during conversion of C. albicans to filamentous form.
In another aspect, the invention includes compositions comprising any of the polypeptides of the invention.
In another aspect, the invention provides purified antibodies that are capable of specifically binding to a polypeptide of the invention. in another aspect, the invention provides a monoclonal antibody capable of specifically binding to a polypeptide of the invention.
In another aspect, the invention provides a method for detecting a polynucleotide from C. albicans in a sample comprising the steps of (a) contacting polynucleotide from C. albicans from a sample with a polynucleotide of this invention under conditions that permit the formation of a stable duplex; and (b) detecting the stable duplex formed in step (a), if any.
The invention also provides a method for detecting a polynucleotide from C. albicans in a sample comprising the steps of (a) conducting an amplification reaction on a polynucleotide in the sample using a primer consisting of a fragment of the polynucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; and (b) detecting the presence of amplified copies of the polynucleotide, if any.
The invention also provides a method for detecting an anti-C. albicans Rbt1 antibody in a biological sample, comprising the steps of: (a) contacting antibody from the sample with a polypeptide of this invention under conditions which permit formation of a stable antigen-antibody complex; and (b) detecting said stable complexes formed in step (a), if any.
The invention also provides a method for detecting a C. albicans Rbt1 polypeptide in a biological sample, comprising the steps of: (a) contacting polypeptide from the biological sample with an antibody of this invention under conditions that permit the formation of a stable antigen-antibody complex; and (b) detecting said stable complexes formed in step (a), if any.
In another aspect, the invention provides methods for identifying an agent that may control virulence in C. albicans. These methods may be in vitro or in vivo (i.e., cell-based). In one embodiment, the invention provides a method for identifying an agent that may control virulence in C. albicans, said method comprising:
(a) contacting at least one agent to be tested with a suitable host cell that has RBT1 function;
(b) analyzing at least one characteristic which is associated with a modulation of RBT1 function in said host cell, wherein an agent is identified by its ability to elicit at least one such characteristic.
In another aspect, the invention provides compositions for controlling virulence in C. albicans comprising any agent identified by the screening methods above.
In another aspect, the invention provides kits for detection or quantification of (a) a polynucleotide comprising an RBT1 polynucleotide from C. albicans; or (b) a C. albicans Rbt1 polypeptide; or (c) an anti-C. albicans Rbt1 antibody in a biological sample. These kits contain (a) a polynucleotide of the invention; or (b) an antibody of the invention; or (c) a polypeptide of the invention, respectively.
In another aspect, the invention provides methods of isolating a polynucleotide sequence from C. albicans that is associated with C. albicans RBT1 function, comprising identifying a transcribed polynucleotide which is up-regulated upon RBT1 expression.
In another aspect, the invention provides methods of inhibiting virulence of C. albicans comprising modulating C. albicans RBT1 function.